|
| Status |
Public on Feb 01, 2016 |
| Title |
2R Sample - repeat 1 - mAdbID:105466 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Pooled mouse T cell Reference Sample
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen and Lymph Nodes
cell type: T cells
sample type: standard reference RNA was derived from pooled mouse T cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extraction Protocol
T cells were suspended in Trizol reagent (Invitrogen/Life Technologies, CA) followed by homogenization by spinning through a Qiashredder column (Qiagen, MD). 0.2 volumes of chloroform was added to the lysate and the aqueous phase separated out after centrifugation. An equal volume of 70% Ethanol was added to this phase and the suspension passed through a RNeasy mini column (Qiagen). Bound RNA was eluted and quantitated. 50ng of total RNA was then amplified using the Ovation RNA amplification system v2 (Nugen, CA) and purified with DNA Clean & Concentrator -25 (Zymo Research).
|
| Label |
cy5
|
| Label protocol |
Agilent ULS Cy5 Labeling Protocol
Total RNA was labeled with Cy5 using the Agilent ULS protocol.
|
| |
|
| Channel 2 |
| Source name |
2R Sample
|
| Organism |
Mus musculus |
| Characteristics |
strain: B10.A, 5C.C7 TCR tg, RAG2-/-
genotype/variation: 5C.C7 TCR tg, RAG2-/-
tissue: Spleen and Lymph Nodes
cell type: T cells
|
| Treatment protocol |
In-vivo treatment: Naïve 5C.C7 T cells were transferred to B10.A, PCC+,CD3epsilon-/- mouse hosts to generate 1R T cells and retransferred to fresh B10.A, PCC+,CD3epsilon-/- hosts to generate 2R T cells. In re-transfer experiments, 1R T cells were enriched by negative selection using a cocktail of antibodies targeting non T cells (B220, CD11b, NK1.1, CD8a and Class II) and then separated using anti-mouse or anti-rat coated Dynabeads (Life Technologies,CA) and magnetic stands. The enriched T cells were sorted to >99% purity on CD4+TCR+ lineage -ve gates using a flow cytometer (BD ARIA II or BD FACS Vantage)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol Extraction Protocol
T cells were suspended in Trizol reagent (Invitrogen/Life Technologies, CA) followed by homogenization by spinning through a Qiashredder column (Qiagen, MD). 0.2 volumes of chloroform was added to the lysate and the aqueous phase separated out after centrifugation. An equal volume of 70% Ethanol was added to this phase and the suspension passed through a RNeasy mini column (Qiagen). Bound RNA was eluted and quantitated. 50ng of total RNA was then amplified using the Ovation RNA amplification system v2 (Nugen, CA) and purified with DNA Clean & Concentrator -25 (Zymo Research).
|
| Label |
cy3
|
| Label protocol |
Agilent ULS Cy3 Labeling Protocol
Total RNA was labeled with Cy3 using the Agilent ULS protocol.
|
| |
|
| |
| Hybridization protocol |
Agilent Hybridization Protocol
According to the manufacture's recommended protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent).
|
| Scan protocol |
Scan_MicronsPerPixelX: 5
Scan_MicronsPerPixelY: 5
Scan_ScannerName: Agilent Technologies Scanner G2505C US45103101
|
| Description |
The sample is RNA from T cells of B10.A, 5C.C7 TCR tg, RAG2-/- which were treated by transferring to B10.A, PCC+,CD3epsilon-/- mouse hosts.
mAdb experiment ID: 105466
|
| Data processing |
Protocol_Name: GE2_105_Dec08 (Read Only)
The samples were analysed using BRBArraytools v.4.2.1. Data was filtered to exclude spots with both intensities below 30. Normalisation was using the lowess smoother
|
| |
|
| Submission date |
Mar 19, 2012 |
| Last update date |
Feb 01, 2016 |
| Contact name |
Nevil John Singh |
| Phone |
3014961236
|
| Fax |
3014960877
|
| Organization name |
NIAID
|
| Department |
LCMI
|
| Lab |
LCMI
|
| Street address |
4 / 211, Center Drive
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL9354 |
| Series (1) |
| GSE36613 |
Density dependent re-tuning of autoreactive T cells alleviates their pathogenicity in a lymphopenic environment |
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